Present in the protein occlusion bodies (OBs) of some baculoviruses is a unique viral-encoded protein which enhances viral infection of the host insect. This protein is referred to herein as the virus enhancing factor (VEF) and/or as the synergistic factor (SF). Pest control Compositions comprising this factor and nuclear polyhedrosis viruses are the subject matter of U.S. Pat. Nos. 4,973,667 & 5,011,685.
Studies on the mode of action of the VEF isolated from Trichoplusia ni (cabbage looper) granulosis virus (TnGV) showed that the VEF caused rapid degradation of the peritrophic membrane which lines the midgut lumen of lepidopterous larvae. Larval bioassays suggested that this alteration made the peritrophic membrane more permeable to invading baculoviruses resulting in at least 25-fold increase in larval mortality (Derksen, A. G. S. and Granados, R. R. Virology 167: 242-250 (1988); and Peters, W. and Wiese, B. J. Insect Physio. 32: 43-49 (1986); both incorporated herein by reference).
The VEF gene was isolated and purified. It comprises a DNA molecule encoding a polypeptide of molecular weight 104 Kd and the protein is found in the granulin fraction of TnGV OBs purified by SEPHACRYL® S200 SUPERFINE (2.6×34 cm) column, possessing a biological activity and wherein said polypeptide has a total of 901 amino acid residues in the amino acid sequence of the polypeptide.
The gene encoding for viral enhancing factor (VEF) of TnGV was cloned from a lambda gtll expression library, and the complete nucleotide sequence determined. The VEF gene encodes a protein with a predicted molecular weight of 104 Kd which does not share homology to any previously reported proteins. The apparent promotor is located 4 bp upstream of the initiation codon and represents a consensus baculovirus late promoter (ATAAG). This has been confirmed by the identification of VEF mRNA in northern blots of infected larvae at 6 days but not 3 days post infection. Three repeats of the sequence ‘TTACAAGA’ which match the baculovirus late promoter in 4 of 5 nucleotide have been identified between 149 and 192 bp upstream of the initiation codon. While the function of these sequences is unknown, they are believed to be transcriptionally active since they diverge from the consensus promoter at the invariant ‘T’ position. Using the VEF gene as a probe in southern blots of genomic DNAs, homologous sequences have been identified in Pseudaletia unipuncta granulosis virus—Hawaiian strain (PuGV-H) and Heliothis armigera granulosis virus (HaGV) but not Erinnyis ello granulosis virus (EeGV) , Autographa californica nuclear polyhedrosis virus (ACMNPV) or Trichoplusia ni nuclear polyhedrosis virus (TnSNPV). In addition, SDS-PAGE analysis of dissolved viral occlusion bodies have demonstrated proteins with a molecular weight similar to VEF in PuGV-H and HaGV.
The gene encoding the synergistic factor (SF) of PuGV-H was cloned and the complete nucleotide sequence determined. The SF gene encodes a protein with a predicted molecular weight of 104Kd which shares a 99.1% and 98.2% homology with the nucleotide and amino acid sequence of the viral enhancing factor (VEF) gene of TNGV, respectively. A majority of the differences in the amino acid sequences of the two viruses result from two reciprocal frameshifts which occur between nucleotide +1962 and +1985 of the SF gene. Both enhancin proteins have similar activity in neonate larvae of Trichoplusia ni (2.4 fold enhancement) and in vitro peritrophic membrane assays. Using a polyclonal antibody directed against TnGV VEF, 17 baculoviruses were screened by western blot hybridization. Cross reactive proteins are found in seven GVs isolated from 4 families of Lepidoptera. These putative enhancing proteins can be separated into 3 groups based on size: HaGV (lloKd); PuGV-H, Pieris rapae granulosis virus (PrGV), Scotogramma trifolii granulosis virus (StGV), and TnGV (104Kd); and Cydia pomonella granulosis virus (CPGV) and Estigmene acrea granulosis virus (80 Kd). The name “enhancin” has been proposed for these enhancing proteins.
Although the genes for some enhancins have been purified and isolated, a need still exists for transgenic plants that have been protected from insects by the introduction of an enhancin gene. Therefore, it is the primary object of the present invention to provide a transgenic plant and a method of protecting a plant by introducing an enhancin gene.